CF patients succumb to pulmonary insufficiency, resulting from complications due to intractable P. aeruginosa biofilms. Because there is no animal model for CF airway disease, in vitro biofilm studies have been performed using a variety of substrata (plastic, glass, stones, etc.) for bacterial attachment and biofilm development. However, unlike abiotic surfaces or even cells, CF airway bacteria are not associated with the epithelium. Instead, they are enmeshed in hypoxic or anaerobic mucopurulent masses in thick mucus lining the airway lumen. Therefore, we hypothesize that P. aeruginosa is undergoing anaerobic respiration (denitrification) during the course of CF airway disease and the process of denitrification is likely critical for optimal growth and survival of the organism in CF airway mucus. Several lines of evidence suggest that these hypotheses are correct. First, antibodies are raised by CF patients against proteins involved in anaerobic metabolism of P. aeruginosa. Second, P. aeruginosa alginate production, that is directly associated with CF morbidity and mortality, and is typically unstable in vitro. Yet it is stabilized during anaerobic growth in the presence of the alternative electron acceptor, nitrate, that is present in ample quantities in CF airway mucus for anaerobic growth of P. aeruginosa. With the completed P. aeruginosa genome and the development of genomics/proteomics, a timely opportunity exists to monitor whole-genome transcription and protein expression patterns under defined conditions that are relevant to the pathogenesis of CF airway disease. The research agenda for this proposal will include a set of experiments using 1- and 2-D gel/MALDI mass spectrometry/proteomics, capillary LCMS mass spectrometry (whole-bacterium proteome analysis), and microarray transcriptional profiling that are organized to fulfill a single primary goal - to identify P. aeruginosa genes and proteins necessary for optimal survival of biofilm bacteria undergoing anaerobic metabolism in mucus derived from CF primary cell lines. The genes/proteins identified in these studies are predicted to mimic those expressed in various stages of CF airway infection. Our LONG TERM GOAL is to identify cellular targets that inhibit or significantly compromise the denitrification process that is occurring by P. aeruginosa in the CF airways.